The overall goal of this project is to identify genes/proteins that play a causal role in brain aging/risk for AD. We will test the hypothesis that interactions of steroids with Ca2+ signaling pathways are underlying causal factors that act in early mid-life, using gene microarrays, combined with sophisticated biostatistical analyses and real-time PCR, protein assays and in situ hybridization. In the first three specific aims, we will extend our previous work by determining which changes in gene expression take place earliest in normal aging rats and also in an animal model of AD, the APP/PS1 mouse. Comparison of these two models will reveal how normal aging transitions into pathological aging and will identify risk factors for AD. In addition, we will carry out microarray analyses of single neurons and gila to that test the hypothesis that aging is a process of neuronal involution and gUallmicroglial activation. We will coordinate this project closely with Project 3, in order to determine the temporal relationship between altered gene expression and Ca 2+ dysregulation assessed by physiological techniques. In the last two specific aims, we will test the hypothesis that steroid hormones and their coordinating transcription factors and Ca2+-regulated signaling molecules play a role in inducing changes in gene expression that mimic those in aging. These studies will be carried out using viral-mediated overexpression/knockdown of steroid hormone receptors or Ca2+-regulated signaling proteins in mixed cultures of hippocampal cells. Subsequent experiments will be carried out in cell-type specific cultures (neurons, gila, microglia) in order to identify where the changes in gene expression originate. Gene expression changes elicited by these treatments will be determined first using microarrays and subsequently, real-time PCR of selected panels of aging biomarkers. Taken together, these studies will elucidate the role of both factors previously implicated in brain aging (steroid hormones, Ca 2+- regulating molecules) and will identify novel factors and pathways, as well. Further, this Project in collaboration with Project 3 and Cores A, B and C will define more clearly the relationship of gene expression cascades to Ca 2+ dysregulation in aging/AD.